animals Article The Use of Stable Isotope Ratio Analysis to Trace European Sea Bass (D. labrax) Originating from Different Farming Systems Francesca Tulli 1 , José M. Moreno-Rojas 2,* , Concetta Maria Messina 3 , Angela Trocino 4 , Gerolamo Xiccato 5 , José M. Muñoz-Redondo 2, Andrea Santulli 3 and Emilio Tibaldi 1 1 Department of Agriculture, Food, Environment and Animal Sciences, University of Udine, Via Sondrio 2, 33100 Udine, Italy; [email protected] (F.T.); [email protected] (E.T.) 2 Department of Food Science and Health, Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA), Alameda del Obispo Centre, Avda. Menendez Pidal, s/n, 14004 Córdoba, Spain; [email protected] 3 Laboratory of Marine Biochemistry and Ecotoxicology, Department of Earth and Sea Science, University of Palermo, Via Barlotta, 4, 91100 Trapani, Italy; [email protected] (C.M.M.); [email protected] (A.S.) 4 Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell’Università 16, 35020 Legnaro, Padova, Italy; [email protected] 5 Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, Viale dell’Università 16, 35020 Legnaro, Padova, Italy; [email protected] * Correspondence: [email protected]; Tel.: +34-671-532758 Received: 21 October 2020; Accepted: 2 November 2020; Published: 5 November 2020 Simple Summary: European sea bass is one of the most economically important fish species in the Mediterranean area. The potential effects of farming systems on the final quality of this product and the recent popular demand for labels to certify the animal rearing origin, which is increasingly used as a marketing tool, have raised the use of analytical techniques that make it possible to differentiate this fish product according to the rearing farming system and authenticate their geographical origin. The aim of this study was to determine whether isotopic ratio mass spectrometry (IRMS) can discriminate farmed European sea bass according to different farming systems (concrete tank inland, sea cages, and extensive methods in valleys or salt works) and geographic origins (different locations scattered throughout Italy). The results of this study showed the viability of δ13C and δ15N to discriminate cultured sea bass from different farming systems (extensive vs. intensive) reared at different geographical sites in Italy. Meanwhile, the measurement of δ18O and δ2H made it possible to distinguish the geographical origin of the sea bass farmed extensively and intensively (in cages). Abstract: This study aimed to determine whether isotopic ratio mass spectrometry (IRMS) can discriminate farmed European sea bass according to different farming systems and geographic origins. Dicentrarchus labrax of commercial size from three different rearing systems (concrete tank inland, sea cages, and extensive methods in valleys or salt works) were collected at the trading period (autumn–winter). For each farming type, different locations spread over Italy were monitored. Once the fish were harvested, the muscle and feed were sampled. For both muscle and feed, δ13C and δ15N were measured by continuous flow elemental analyzer isotope ratio mass spectrometry (CF-EA-IRMS) with the goal of discriminating samples based on the rearing system. Additional δ2H and δ18O measurements of fish samples were performed by continuous flow total combustion elemental analyzer isotope ratio mass spectrometry (CF-TC/EA-IRMS) to track the geographical origin. The measurements of δ13C and δ15N made it possible to discriminate cultured sea bass from different farming systems (extensive vs. intensive) reared at different geographical sites in Italy. Additional information was obtained from δ18O and δ2H, which enabled the geographical areas of origin of the sea bass farmed extensively and intensively (in cages) to be distinguished. Animals 2020, 10, 2042; doi:10.3390/ani10112042 www.mdpi.com/journal/animals Animals 2020, 10, 2042 2 of 15 Keywords: aquaculture; Dicentrarchus labrax; stable isotopes; traceability; farming system; geographic origin; IRMS; sea bass; fish; authentication 1. Introduction In recent years, the trade of seafood has experienced strong growth due to the rising world population and increases in the annual consumption per capita of seafood from 9.9 kg in the 1960s to over 20 kg in 2013 [1]. In 2013, fish accounted for 17% of the global population’s intake of animal protein and 6.7% of all protein consumed [1]. Consequently, international trade of fish products has addressed the evolution of food safety and food quality issues as emphasized by several EU Directives introduced into the chain for fisheries and aquaculture products with the concept “from farm to fork” usually based on the Codex Alimentarius provisions. In addition, national and transnational regulations regarding meat traceability have been imposed (European Regulation (EU) n. 1379/2013) to ensure more accessible details for retailers and consumers about labeling, packaging, and origin of wild capture and aquaculture products. In this respect, consumers are more and more concerned about the origin of foodstuffs for both health and ecological reasons, and food quality has become an essential parameter for their consumer preferences. Therefore, product differentiation appears to be a fundamental issue for the further development of the fish farming industry distributed around the whole Mediterranean area involved in different rearing and environmental systems. The potential effects of different fish farming systems involve modifications to the final product quality such as fish appearance, organoleptic properties or even nutritional characteristics [2]. Feeding and breeding conditions are known to affect the nutritional quality of the fillet, the high content in essential polyunsaturated omega 3 and omega 6 fatty acids of wild fish and suitably fed farmed fish being of note [3]. Linked to, but different from, consumers’ demand for fish quality standards is the public perception that aquaculture can harm the environment [1]. In recent years, intensive farming has led to misperceptions and mistrust among consumers. Thus, authorities have acknowledged the need for labels that certify animal health and welfare, food safety and quality, environmental integrity, and social responsibility associated with aquaculture. The aims of such labels are (i) to reassure producers, buyers, consumers and civil society regarding the quality and safety of aquaculture products, and (ii) to provide a further tool to support responsible and sustainable aquaculture. A popular demand for labels to certify the animal rearing origin has emerged over the last years since, and, according to formal regulation, it is increasingly used as a marketing tool rather than a designation of quality and safety. Thus, besides being a tool to guarantee food safety, tracking, and traceability, these labels are also of major interest to retail business as a powerful communication tool aimed at improving consumer confidence [4–10]. However, food products with geographical indications and designation of origins following the European Regulation (EU) n. 1151/2012 are generally expensive but bring greater benefits to the producers than ordinary products. In this sense, consumers seem to be prepared to accept higher prices linked to superior quality, certification of the production process and the product origin. European sea bass (Dicentrarchus labrax L.) is one of the most economically important fish species in the Mediterranean area [11] as it is one of the most requested marine species for its overall quality, with an interesting polyunsaturated fatty acid (PUFA) content 0.58 g/100 g of docosahexaenoic acid (DHA, 22:6(n 3)) and 0.44 g/100 g of eicosapentaenoic acid (EPA, 20:5(n 3)) (EC, 2015 [12]). Sea bass − − is found in the Mediterranean area in a variety of different culture systems including highly intensive recirculating systems, flow through concrete raceways or pond systems, floating sea cages, as well as traditional extensive pond systems located in highly environmentally sensitive areas [2]. Currently, intensive systems are the most common rearing method in this area due to a higher production yield [13]. Many publications have considered the authentication and quality of sea bass in relation to different factors, such as eco-physiological factors, diet, rearing condition, and differences between Animals 2020, 10, 2042 3 of 15 wild and farmed fish, but the reported results refer to a limited period of time, a restricted geographical area, or to only one type of culture system compared to the wild [2,14–23]. Despite the attention given to these issues, mislabeling, whether accidental or fraudulent, is expected to occur [24]. In the last few years, an increase in such mislabeling concerning the product processing and fish origin has been reported [25]. Therefore, new tools enabling simple and accurate discrimination between farming systems and authenticating their geographical provenance would be extremely valuable. Stable isotope ratio mass spectrometry (S-IRMS) could be used as an alternative tool to PCR-DGGE techniques that have been recently proposed to indirectly discriminate the geographical origin of fish by the analysis of the DNA fragments of microorganisms [26]. The isotopic content of an animal’s diet is known to affect the isotopic ratio of its meat [27–29]. For this reason, IRMS has previously been used for ecosystem studies